Antibacterial compositions and methods

ABSTRACT

Imines of 2-formylquinoxaline-3-carboxylic acid-1,4-dioxides and their salts are obtained through treatment of the lactone or a salt of 2-dihydroxymethylquinoxaline-N,N-dioxide-3-carboxylic acid with a reactant bearing a free primary amino group. The resultant compounds and their non-toxic salts are antibacterial agents and can be incorporated in pharmaceutical compositions and feedstuffs for this use. A typical embodiment is 2(carbomethoxyhydrazonomethyl)-quinoxaline-3-carboxylic acid-1,4dioxide.

United States Patent Seng et al. Jan. 14, 1975 1 ANTIBACTERIALCOMPOSITIONS AND [56] References Cited METHODS UNITED STATES PATENTS[75] Inventors: Florin Seng, Cologne-Buchheim; 3,371,090 2/1968 Johnston260/240G Km odemhal'Globuschi FORElGN PATENTS OR APPLICATIONS Karl GeorgMetzger, Wuppertal- 7 Elberfald, a of Germany 1,215,815 12/1970 GreatBritain 260/-50 R [73] Assignee: Bayer Akfiengeseuschaft PrimaryExaminer-leron1e D. Goldberg Leverkusen, Germany [22] Filed: Aug. 20,1973 57] CT [2]] Appl. No.: 389,885 Imines of2-formylquinoxaline-3-carboxylic acid-l,4- dioxides and their salts areobtained through treat- Related Apphcatmn Data ment of the lactone or asalt of 2- [62] P' PI 323,953 1973 wh'chdihydroxymethylquinoxaline-N,N-dioxide-3-carboxylic 1s a division ofSer. No. 130,007, March 31, 1971. acid with a reactant bearing a freeprimary amino group. The resultant compounds and their non-toxic [30]Fol-Hg Apphcatlon Pnomy Data salts are antibacterial agents and can beincorporated P 2, 1970 Germany 2015676 in pharmaceutical compositionsand feedstuffs for this use. A typical embodiment is 2- U-S. Cltarbomethoxyhydra onomethy]) quinoxaline 3- Int. Clcarboxylic acid l4-dioxide [58] Field of Search 424/250, 323, 953

12 Claims, No Drawings ANTIBACTERIAL COMPOSITIONS AND METHODS This is adivision of application Scr. No. 323,953 filed Jan. 15, I973, which is adivisional of Ser. No. 130,007 filed Mar. 31, I971.

The present invention relates to new imines of 2-formyl-quinoxaline-3-carboxylic acid-l,4dioxides and their salts, toprocesses for their preparation, to the use of the new compounds asmedicaments in human medicine and veterinary medicine, to their use asfeedstuff additives, especially in raising young animals or fatstock,and to compositions adapted to this use.

The new imines and salts have the general formula:

\ CODY l CH=NR wherein Y is hydrogen, an alkali metal cation or thecation and each of R and R is identical to or different from the otherand is selected from the group consisting a. alkyl, substituted alkyl orcycloalkyl;

in which each of R and R when taken indepen-i dently is identical to ordifferent from the other, and is selected from the group consisting ofhy drogen, alkyl or substituted alkyl, or when R and R are takentogether with the nitrogen atom to which they are attached a 5- to7-membered heterocyclic ring optionally containing as a ring memberoxygen, sulphur, S0 or N-alkyl;

in which X is O, S or NH and, R and R are as above defined;

in which R is alkyl or substituted alkyl;

NIIER in which R is phenyl, pyridyl or norbornyl, and X is as definedabove; f.

in which R, R and X are as defined above; or

in which each of R and R when taken independently is identical to ordifferent from the other and is selected from the group consisting ofhydrogen, alkyl or hydroxyalkyl of from 1 to 4 carbon atoms, or when Rand R are taken together with the nitrogen atom to which they areattached, a o-membered heterocyclic ring optionally containing as a ringmember oxygen or S0 in which X is O, S or NH, and R and R are as hereindefined;

in which R is alkyl or hydroxyalkyl of from 1 to 4 carbon atoms; and

NH -R in which R is phenyl, pyridyl or norbornyl, and X 5 is as hereindefined.

Alphatic groups embraced by R and R include straight-chain or branchedalkyl groups of from l to 6,

preferably 1 to 4, carbon atoms. Cycloaliphatic radicals 10 Thesubstituents R and R are hydrogen or alkyl of from 1 to 4, preferably 1or 2, carbon atoms. These alkyl groups can be optionally substitutedwith hydroxy, alkoxy or acyloxy, alkoxy and acyloxy groupsv containing 1to 4, preferably 1 or 2, carbon atoms. Thus included are ethyl,n-propyl, isopropyl, n-butyl, isobutyl and tert.-butyl, as well as thecorresponding groups substituted by hydroxy.

R and R when taken together with the nitrogen atom to which they areattached, can also form a heterocyclic ring, preferably containing 6ring members, and preferably with an oxygen atom, a sulphur atom, an N-alkyl group containing 1 to 4, preferably 1 or 2, carbon atoms, or theSO, group, as a ring member in the pposition relative to the nitrogenatom to which R and R are attached.

R is an alkyl of from I to 4, preferably 1 or 2, carbon atoms which mayalso be optionally substituted by hydroxy, alkoxy, acyloxy, alkoxy andacyloxy containing 1 to 4, preferably 1 or 2, carbon atoms. The hydroxygroup is a particularly preferred substituent. R thus embraces suchgroups as methyl, ethyl, 2-hydroxyethyl and the like.

The alkali metal cation Y is, for example, that of sodium or potassium,preferably that of sodium. I

R is phenyl, pyridyl or norborn-2-yl. When R is pyridyl, it can bebonded in the 2-, 3- or 4-position relative to the pyridyl nitrogenatom.

The above class of imines are obtained according to the process of thepresent invention by treatment of loxo-3-hydroxyl ,3-dihydro-furo-(3,4-b)-quinoxaline- 4,9-dioxide, which has the formula:

or a salt thereof of the formula i M II; i O. x

with an amine of the general formula H N-R, in which R is as previouslydefined, M is an alkali metal or alkaline earth metal cation and x is lor 2.

The salts of the formula can be obtained from the lactone of formulathrough treatment with alkali metal or alkaline earth metal hydrogencarbonates.

M is preferably the cation of sodium, potassium or most preferablycalcium.

Both inorganic and organic polar solvents can be used as diluents forthe reaction according to the invention, such as for example water,lower aliphatic alcohols of l to 4 carbon atoms, lower aliphaticnitriles such as acetonitrile, tetrahydrofurane, dioxane,dimethoxyethane, pyridine, dimethylformamide and the like.

The reaction according to the invention is carried out at a temperatureof about 0C to about 50C, preferably 20C to about 35C.

In practice, the lactone or lactone salts are dissolved or suspended inthe diluent, and this solution or suspension is then treated with anappropriate quantity of the amine. The formation of the imine or of theimine salt takes placein a weakly exothermic reaction and the finalproduct is then isolated through conventional methods.

The imine salts (1) can also be prepared by the reaction of the freeacids with amines.

The salts may be obtained in a subsequent step by conventionaltechniques or directly in'the reaction of the lactone with the amine. lfabout 2 mols of the amine per mol of lactone are employed, the productwill be in the form of that amine salt. If an alkali metal salt oralkaline earth metal salt of the lactone is employed, or if it isdesired to obtain the free acid from the lactone, only about 1 mol ofthe amine per mol of the lactone or salt is required.

The course of the process according to the invention can thus beillustrated by the following equations:

OONa 3 H +H Cl-l 4 The following examples will serve to further typifythe nature of this invention without being a limitation on the scopethereof.

EXAMPLE 1 23.4 g (0.1 mol) of l-oxo-3-hydroxy-l,3- 30dihydrofuro-(3,4-b)-quinoxaline-4,9-dioxide are suspended in 60 ml ofwater and G (0.2 mol) of 60% strength aqueous isopropylamine solutionare added. The temperature is kept below C by slight cooling. After afew minutes, a clear solution is produced. Evaporation in vacuo yields45 g of the compound of the for mula i C0OH H (I? H3 in the form ofyellow crystals, which after recrystallisation from isopropanol melt at123-25C, with decomposition.

hydroxymethyl)-3-carboxylic acid-quinoxaline-di-N- oxide are dissolvedin 100 ml of water and 7.3 g (0.1 mol) of tert.-butylamine are added.The temperature is kept at 25C by cooling. After 1 hour, the solution isevaporated in vacuo and 32 g of the compound of the formula u OONa H ca:(L-cu I N\ COONa I H3 l C1-t==l CH Analysis: C H N NaO (molecular weight3! l) Calculated: C 54.0% H 4.5% N 13.5% Na 7.4% Found: 53.7% H 4.9% Nl3.5% Na 6.9%

The l-oxo-3-hydroxy-l ,3-dihydro-furo( 3,4-b quinoxaline-4,9-dioxide ofthe formula (I) required as the starting compound can be obtained asfollows:

30.7 g (0.1 mol) of 2-bismethoxy-methyl-3-dimethylaminocarbonyl-quinoxaline-l ,4-di-N-oxide are introduced into mlof 10% strength aqueous hydrochloric acid. A clear solution results, andafter a short time the compound according to the invention separates outin the form of a yellow precipitate, which is filtered off after 6hours. 17 g (72.6% of theory) of l-oxo-3-hydroxyl ,3-dihydro-furo-( 3,4-b )-quinoxaline-4,9-dioxide are thus obtained in the form of yellowcrystals.

The compound is purified by dissolving it in sodium bicarbonatesolution, filtering and acidifying the filtrate. The purified compoundmelts at l56-l59C, whilst foaming.

Analysis: C,.,H N,O,(235) Calculated: C 5l.3% H 2.6% N 12.0% Found: C52.0% H 2.8% N [2.6%

The alkali metal salts or alkaline earth metal salts of 1-oxo-3-hydroxyl,3-dihydro-fur0-( 3 ,4-b)quin0xaline- 4,9-dioxide can be manufactured asfollows:

The lactone (9) is suspended in water and approximately thestoichiometrically required amount of the 5 alkali metal hydrogencarbonate or alkaline earth metal Example Melting Point: (C) No.Compound (decomposition) 3 143-44 00 H NCH 3 3 (9 0O H N-C H \NL CH=N-CH O ff G3 ()Ol'I N-C l*l CFN-C H l 3 7 n 6 9 e v5 138-40 COOH N-- -CH 33 CH I i CH==N {CH 1 e COOH.

15 16 Example Melting Point: (C) No. Compound (decomposition) OONaca=nnuc Q? k OONa If CH==N-NHI.

COOH CH==N-Nl-!COC H As has already been mentioned, the new compounds ofthe invention surprisingly show an excellent chemotherapeutic activity.Their chemotherapeutic action was examined both in animal experiments(oral and subcutaneous administration) with acute bacterial infections,and in vitro. In both cases the compounds show a very good antibacterialaction, and the range of action encompasses both Gram-negative andGrampositive bacteria. The chemotherapeutic activity of the compoundsaccording to the invention permits their use in human medicine and inveterinary medicine. Furthermore, the compounds can be employed asfeedstuff additives, especially in raising young animals or fatstock.The good in vitro and in vivo activity of the compounds according to theinvention can be seen from Tables 1. 2 and 3 below.

R HHCOOCH CH OH The minimum inhibitory concentrations in vitro for someof the new compounds shown in Table l (MIC) were determined by the platetest in an agar medium of the following composition:

Readings were taken after 24 and 48 hours, and the'incubationtemperature was about 37C.

MIC in y/ml of medium For the compound of Example 3, the followingminimal inhibitory concentrations (MIC) (y/ml of nutrient medium) weremeasured by the series dilution test (PPLO medium), incubationtemperature 37C, deter- Compound of Compound of Compound of BacteriumExample IO Example 12 Example 13 mmatlon after 24 and 48 hoursEscherichia coli A 26] 20 Escherichia coli C I65 50 Imieim vulguri'sBacterium MIC species 150 ll) Pxcudumunus Mycoplasma galli'replic'uniI00 s' do. grunularum 25 Bonn I00 H10 do. huvi'rhi'm'x 200 Isz'iulumimasucruginuxa WEIIICI' 100 Klehsiella pneumonia 63 I00 much-e11 In animalexperiments on mice, the effective 100% ;7: 3085 dose (ED in mg/Kg wasdetermined for certain com- L 1 (ICOCCHS 3x m 20 pounds of the inventionafter intraperitoneal infection p and subcutaneous (s.c.) or oral (p.o.)administration of pyogem the preparation.

Table 3 Compound of Compound of Compound Compound of of Example 3Example 8 Example l3 Example 14 Bacterium 8.6. 13.67 543. p.C. SVC. PAC.S.C. [3.6.

Escherichia 50 I00 50 I00 25 50 I00 coli C I65 Staphylococ- 25 50 CUSBUI'BUS TABLE 2 In general, it has proved advantageous, in acute gen- 35eral infections, to administer amounts of about 5 mg to gi gi i p z i z.S i about 200 mg per kilogram, preferably about 25 to i g y t e series Ig p 6 about 50 mg per kilogram of body weight per day, to z g f g z h eermma' achieve effective results. Nevertheless it can at times be O t ea ter an necessary to deviate from the amounts mentioned, in

40 particular depending on the body weight of the test ani- Compound ofmal or patient or on the nature of the method of admin- BacteriumExample 13 istration, but also because of the type of animal and its S,I f r ATCC 9700 50 individual behaviour towards the medicament, or be-OLOCCMS CH S p 8564 100 cause of the nature of the formulation of thelatter, and do. 8580 50 the point in time or interval at whichadministration 3: 323g :88 takes place. Thus it can, in some cases,suffice to use d0. 87H less than the abovementioned minimum amount,whilst slrepmvvcvus faewlis ATCC 100 in other cases the upper limitmentioned must be ex- 33 3 ceeded. In the case of the administration oflarger do. species mo 50 amounts it can be advisable to divide theseinto several 38- gggg- 32 individual doses over the course of the day.The same gmhemhm C I65 50400 range of dosages is envisaged foradministration in g B g g human medicine. The other comments made above8: 4 t 5 also apply in a general sense. do. A 26! 25-50 5 5 Accordingly,the present invention provides a pharpmlmdsz'imbms 5 1% maceuticalcomposition containing as an active ingredo. 2935 12 dient at least oneof the new compounds of the general 38- 3" m i? formula (1) given abovein admixture with a pharmapseuanm n Mm'gmm w 400 ceutically acceptablesolid or liquid diluent or carrier 38- g l as hereinafter defined.Klebsielh, 'ATCC [003, In the present specification the expressionpharmago. 6K3l0 g8 ceutically acceptable diluent or carrier means a nongpamwphii B8 n '2 toxic substance that when mixed with the active ingre-(vrynebacierium diphleriae' gravis 5l0 dient or ingredients renders itsuitable for administra- SJ #3 I, tion. The expression preferablyexcludes water and dov BRL l2 low-molecular weight organic solventscommonly used l 6 in chemical synthesis, except in the presence of otherM i'mplusmu gulliri'piiz'um 6 h l Mywplusnn, gum-WWW) 6 p armaceutica ynecessary ingredients such as sa ts in do, g 3 correct quantities torender the composition isotonic, M ywhacierium tuberculosis H 37 RV 40'lmcusiircil in ii PPLO medium buffers, surfactants, colouring andflavouring agents, and preservatives. Examples of suitable liquiddiluents and carriers are vegetable oils, glycerol, propylene glycol,polyols, buffered aqueous solutions, isotonic saline aqueous solutions,syrups and lotion bases. Examples of suitable solid diluents andcarriers are starches, cellulose and its derivatives, sugars, stearatesand stearic acid, talc, and ointment bases. Examples of pharmaceuticalcompositions according to the invention are ointments, pastes, creams,sprays, lotions, aqueous and non-aqueous suspensions, emulsions, andsolutions (including parenterally injectable solutions), elixirs andsyrups, and granulates and powders either free-flowing or compressedinto tablets.

Pharmaceutical compositions of the invention adapted for oraladministration are a preferred embodiment of the invention. The diluentsand carriers used are preferably therefore those that adapt the activeingredient or ingredients for oral administration. Examples of suchdiluents and carriers are solid vehicles, excipients and lubricants suchas glucose, lactose and sucrose, corn and potato starch, sodiumcarboxymethylcellulose, ethyl cellulose and cellulose acetate, powderedgum tragacanth, gelatin, alginic acid, agar, talc, stearic acid andsodium, calcium and magnesium stearates, sodium lauryl sulphate,polyvinyl-pyrrolidone, sodium citrate, calcium carbonate, and dicalciumphosphate.

The pharmaceutical compositions of the invention may also contain othernon-toxic adjuvants and modifiers such as dyes, surfactants, perfumes,flavouring agents, such as sweeteners, preservatives and biocides.

Pharmaceutical compositions of the invention adapted for parenteralinjection are another preferred embodiment of the invention. Thediluents and carriers used are therefore preferably those that adapt theactive ingredient for parenteral administration. Examples of diluentsand carriers that adapt the active ingredient for parenteraladministration are solvents and suspending diluents such as water,vegetable fatty oils, such as sesame oil, groundnut oil, corn oil, andcottonseed oil, aqueous propylene glycol, N,N'-dimethylformamide, anddimethyl sulphoxide. In general, any non-aqueous diluent can be usedthat does not reduce the activity of the active ingredient and isnon-toxic in the dose employed.

For the administration of the water-soluble compounds of the inventionby parenteral injection sterile aqueous solutions can be employed, andare within the scope of the pharmaceutical compositions of theinvention. Such aqueous solutions should preferably when necessary bebuffered in the usual manner, and the liquid diluent should preferablybefore administration be rendered isotonic by adding the requisiteamount of salt or glucose. Such sterile buffered isotonic solutions areespecially suitable for intravenous, intramuscular and intraperitonealinjections. These pharmaceutical compositions of the invention canfurther contain local anaesthetics or substances that promote thediffusion of the active ingredient, for example hyaluronidase.

The pharmaceutical compositions of the invention preferably contain 0.5to 90 wt.% of at least one new compound of the invention.

The present invention also provides medicaments in dosage unit form ashereinafter defined comprising as an active ingredient at least onecompound of general formula (1) given above either along or in admixturewith a pharmaceutically acceptable solid or liquid diluent or carrier.In this case the diluent or carrier is preferably as defined above butcan also be water or another common solvent.

The expression medicament in dosage unit form as used in the presentspecification means a medicament in the form of discrete portions eachcontaining a unit dose or a multiple or sub-multiple of a unit dose ofthe active ingredients(s); for example, one, two, three or four unitdoses or a half, a third or a quarter of a unit dose. A unit dose" isthe amount of the active ingredient(s) to be administered on oneoccasion and will usually be a daily dose, or for example a half, athird, or a quarter of a daily dose depending on whether the medicamentis to be administered once or, for example, twice, 3 times, or 4 times aday.

The discrete portions constituting the medicament in dosage unit formcan include a protective envelope. The active ingredient can beundiluted and contained in such an envelope, or can be mixed with apharmaceutically acceptable solid or liquid diluent or carrier asdefined above. Such portions can for example be in monolithic coherentform, such as tablets, lozenges, pastilles, pills, suppositories, ordragees; in wrapped or concealed form, the active ingredients beingwithin a protective envelope, such as wrapped powders, cachets, sachets,capsules, or ampoules; or in the form of a sterile solution suitable forparenteral injection, such as ampoules of buffered, isotonic, sterile,pyrogenfree aqueous solution; or in any other form known in the art.

As stated above, peroral administration is a preferred mode ofadministration. Preferred medicaments in dosage unit form according tothe invention are therefore those adapted for oral administration, suchas tablets, pills, dragees, capsules, and cachets, as well as wrappedpowders containing the active ingredient in powdered form with apowdered diluent or carrier for suspension in water before being taken.

As also stated above'a further preferred mode of administration isparenteral administration. Preferred medicaments in dosage unitformaccording to the invention are therefore those adapted for parenteralinjection, such as ampoules containing a measured quantity of a sterileisotonic saline injectable aqueous solution of .the new activeingredient, which may be buffered with a pharmaceutically acceptablebuffer and are preferably free of pyrogens.

The preferred unit dose for administration of the medicaments of theinvention is 250-16000 mg. of active ingredient, preferably 1250-4000mg. This will usually be administered once daily.

The invention further provides a method of combatting bacterialinfection in an animal which comprises administering to the animal.(preferably parent'erally or perorally) an effectiveamount of one ofthe new compounds, either alone, as a pharmaceutical compositionaccording to the invention, or as a medicament in dosage unit formaccording to the invention.

22 i I COONa H i @N CkkNl-l l I. i

7. A method of treating bacterial infections in humans and animals whichcomprises administering to said human or animal an antibacteriallyeffective amount of a compound of the formula cause considerableeconomic damage and which can be prevented or treated by administeringthe new compounds in the feedstuff or in the drinking water, there maybe mentioned, in addition to general infections, infection of the airsac in chicks, and mastitis in cows.

What is claimed is: 1. An antibacterial composition which comprises anantibacterially effective amount of a compound of the i. (I iad formulaor pharmaceutically acceptable non-toxic salt thereof 25 wherein N OOY Yis hydrogen, an alkali metal cation or the cation CB== N-R o and each ofR and R is or a pharmaceutically acceptable non-toxic salt thereof Twherein H N Y is hydrogen, an alkali metal cation or the cation 5 (B-NH-C RNH L 40 and each of R and R is in combination with apharmaceutically acceptable 0 non-toxic inert diluent or carrier. l 8. Amethod according to claim 7 wherein Y is hy- H N N drogen.

2 9. A method according to claim 7 wherein Y is a sodium or potassiumcation.

10. A method according to claim 7 wherein the administration is oral.11. A method according to claim 7 wherein the administration is bysubcutaneous injection.

12. The method according to claim 7 wherein the compound is incombination with a pharmaceutically acceptable non-toxic inert diluentor carrier.

2. An antibacterial composition according to claim 1 wherein Y ishydrogen. 5 2 3. An antibacterial composition according to claim 1 Nwherein Y is a sodium or potassium cation.

coosa 4. An antibacterial composition according to claim 1 I in oraladministration form. cps- 5. An antibacterial composition according toclaim 1 w g in subcutaneous administration form. 6

wherein the compound is 6. An antibacterial composition according toclaim 1

2. An antibacterial composition according to claim 1 wherein Y ishydrogen.
 3. An antibacterial composition according to claim 1 wherein Yis a sodium or potassium cation.
 4. An antibacterial compositionaccording to claim 1 in oral administration form.
 5. An antibacterialcomposition according to claim 1 in subcutaneous administration form. 6.An antibacterial composition according to claim 1 wherein the compoundis
 7. A method of treating bacterial infections in humans and animalswhich comprises administering to said human or animal an antibacteriallyeffective amount of a compound of the formula
 8. A method according toclaim 7 wherein Y is hydrogen.
 9. A method according to claim 7 whereinY is a sodium or potassium cation.
 10. A method according to claim 7wherein the administration is oral.
 11. A method according to claim 7wherein the administration is by subcutaneous injection.
 12. The methodaccording to claim 7 wherein the compound is